PROJECT SUMMARY/ABSTRACT Understanding the molecular basis for how hosts defend against viruses is an essential requirement for future development of antiviral therapeutics. Members of the poly ADP ribose polymerase (PARP) family display hallmarks typical of antiviral defense proteins including interferon-inducibility, accelerated evolution, and the ability to inhibit virus infection. A defining feature of PARPs is the enzymatic activity of ADP-ribosylation. Despite the imperative for understanding the mechanisms of the antiviral response, there is almost nothing known about what activates PARP enzymatic activity during the antiviral response, nor how this activity promotes an antiviral state. There is a critical need to fill in this gap because understanding the PARP- mediated antiviral response opens up new strategies of pharmacological intervention for cytokine-driven and viral diseases. Our long-term goal is to define the mechanisms by which viral and host non-protein-coding-RNA (ncRNA) regulates virus infection and disease. Consistent with this goal, our overall objective here is to determine how RNase L, a canonical component of the antiviral response, performs the newly described activity of generating ncRNA that induces PARP activity and how this increased PARP activity is antiviral. Our central hypothesis is that ncRNA generated by RNase L activity increases PARP activity and creates a heightened antiviral state by post-transcriptional mechanisms. Our hypothesis is formulated on our preliminary data demonstrating that RNase L activity is both necessary and sufficient to induce a diffusible PARP- dependent antiviral factor. The rationale for this proposed research is that understanding how RNase L products activate antiviral PARPs may identify critical new targets for cytokine-driven and viral diseases, as well as improve our overall understanding of three established/emerging areas of the antiviral response: RNase L, ncRNA biology and PARPs. We plan to test our central hypothesis and complete the objectives outlined in this proposal via the following two specific aims: 1) Determine how RNase L induces antiviral ADP-ribosylation, and 2) Determine how PARP activity creates an antiviral state. Our contribution here is expected to be a detailed understanding of how ADP-ribosylation is generated by RNase L-product ncRNA and how this contributes to the antiviral response. This contribution will be significant because it is expected to have translational importance in the prevention and treatment of a range of cytokine-driven diseases. The research proposed in this application is innovative, in our opinion, because it represents a new and substantive departure from the status quo by focusing on the modulation of PARP activity as a means to alter antiviral response and cytokine levels.